Micro-fluidic chip and testing method thereof

Abstract

The present disclosure relates to a micro-fluidic chip, comprising a substrate; a cover piece, the cover piece comprising a groove extending in the length direction; and a micro-channel defined by the substrate and the groove, wherein the groove of the cover piece comprises a channel area, the channel area is sequentially provided with a sample adding area, a reaction area and a control area in the length direction, and the sample adding area, the reaction area and the control area are in fluid communication with each other, wherein a control valve is arranged in the control area, the control valve is configured to control the flow of liquid in the reaction area to the control area, the height of the reaction area is lower than that of the control area, and the width of the reaction area is smaller than the width of the control area. The control valve can enable the liquid in the reaction area to flow towards the control area at the required time, so that the time in the sample reaction area is controlled, and the liquid flows into the control area after full reaction and is not retained in a channel of the reaction area.

Description

A microfluidic chip and its detection method

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202311230967.4, entitled "A Microfluidic Chip", and Chinese Patent Application No. 202311230921.2, entitled "A Microfluidic Chip and a Detection Method Thereof", both filed on September 22, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of in vitro diagnostic technology, specifically to a microfluidic chip and its detection method. Background Technology

[0004] Microfluidic chips are the primary platform for realizing microfluidic technology, integrating basic operational units such as sample preparation, reaction, separation, and detection in biological, chemical, and medical analytical processes onto a tiny chip. They automate the entire analytical process through microchannels, enabling various functions of conventional chemical or biological laboratories. Microfluidic chips offer advantages such as compact size, small sample and reagent usage, rapid reaction speed, large-scale parallel processing, and disposable operation, demonstrating enormous potential in biology, chemistry, and medicine. In recent years, they have developed into a new interdisciplinary research field encompassing biology, chemistry, medicine, fluid dynamics, electronics, materials science, and mechanics.

[0005] Microfluidic chips typically incorporate a fixed volume of reaction solution. Utilizing their unique structure, the sample flows through microchannels via capillary action after being added, eventually reaching the waste liquid zone to complete the automated reaction process. This process is then combined with an optical analyzer for detection, allowing for analysis of the results.

[0006] In known microfluidic chips, the sample loading well and buffer well are usually located upstream of the labeling region, which in turn is located upstream of the detection region. Therefore, after the sample passes through the sample loading well, it will first react in the labeling region and then enter the detection region. For highly viscous samples, they cannot propel the signal molecules in the labeling region to the detection region on their own, so a subsequent buffer solution is needed to propel them. However, this method will dilute the sample, causing inaccurate detection results. At the same time, other substances contained in the sample may also interfere with the signal molecules in the labeling region.

[0007] The flow and control of samples within microfluidic chips is a key concern. One known microfluidic chip with controllable reaction time involves embedding a water-absorbing material at the sample outlet. This material contacts the microchannels, adsorbing the sample retained within. However, this method cannot control the timing of sample adsorption, potentially leading to incomplete reactions within the channels.

[0008] Another known microfluidic chip includes a microchannel with a defined height and width formed by a substrate and a cover plate. Sample fluid can flow to the outlet of the microchannel under the capillary force of the microchannel. The height of the top wall of the microchannel is lower than the height of the contact surface between the substrate and the cover plate. However, this method requires ensuring the injection volume of sample liquid, and leakage may still occur during operation, making the detection results inaccurate.

[0009] Summary of the Invention

[0010] The purpose of this invention is to address the shortcomings of existing technologies by providing a microfluidic chip and its detection method, thereby solving the technical problems mentioned in the background section.

[0011] On the one hand, to achieve the above objectives, the present invention proposes a microfluidic chip, including a substrate, a cover plate, and a microchannel formed by the substrate and the cover plate. The microchannel includes a buffer loading area, a reaction area, and a control area, which are sequentially connected along the length direction. A movable absorbent material is disposed in the control area, which can contact or move away from the reaction area. The reaction area includes a labeling area, a detection area, and a sample loading area, and the detection area and the sample loading area are both located on the same side of the labeling area.

[0012] In some examples, the cover sheet has a groove along its length, which, together with the substrate, forms the microchannel.

[0013] In some examples, the detection area is provided with detection sites and quality control sites sequentially along its length, and the number of detection sites and quality control sites is one or more.

[0014] In some examples, the labeled region includes an antibody / antigen labeled with a signal molecule, the detection site of the detection region is coated with an antibody / antigen capable of binding to a target analyte, the target analyte can simultaneously bind to the antibody / antigen of the labeled region and the detection site to form a dual antigen / antibody sandwich complex, and the quality control site of the detection region is coated with an antibody / antigen capable of binding to the antibody / antigen labeled with the signal molecule of the labeled region.

[0015] In some examples, a braking hole is provided on the side of the control area opposite to the reaction area. The braking hole is a rectangular through-hole that penetrates the substrate and the cover plate and extends at least partially into the absorbent material in the control area. The absorbent material has a through-hole at the end away from the reaction area, and the through-hole is located within the braking hole.

[0016] In some examples, the buffer solution loading area is provided with a buffer solution well that connects the upper and lower surfaces of the cover plate, and the sample loading area is provided with a sample loading well that connects the upper and lower surfaces of the cover plate.

[0017] In some examples, the sample application area and the marking area are respectively located on both sides of the detection area.

[0018] In some examples, the sample application area is located between the marking area and the detection area.

[0019] In some examples, the signaling molecule-labeled antigen / antibody is a signaling molecule-labeled antigen / antibody molecule.

[0020] On the other hand, the present invention also proposes a method for detecting microfluidic chips, which uses the microfluidic chip as described above for detection, and the method includes at least the following steps:

[0021] The sample is added into the microchannel through the sample addition area, and the absorbent material is kept away from the reaction area.

[0022] Once the sample flows backward through the detection zone under capillary action and meets the first judgment condition, the absorbent material is moved to contact the reaction zone to absorb excess sample until the flow stops, and the sample remaining in the microchannel reacts with the detection zone; then, the absorbent material either detaches from or continues to contact the reaction zone.

[0023] After the second reaction time period, the buffer solution is added to the microchannel through the buffer sample loading area. The buffer solution, under capillary action, passes through the labeling area, washing away the antigen / antibody coated with the signal molecules in the labeling area before entering the detection area. Once the signal molecules in the reaction area channel have combined with the sample, the absorbent material in the moving control area contacts the reaction area, adsorbing any excess sample into the control area.

[0024] After the excess sample in the channel is absorbed by the absorbent material and the signal molecules in the labeled area dissolve and fill the reaction zone channel, move the absorbent material away from the reaction zone to break the contact between the absorbent material and the reaction zone channel.

[0025] After the third reaction time period, the absorbent material in the moving control area comes into contact with the detection area, and the excess liquid, including signal molecules, is adsorbed into the control area by the absorbent material.

[0026] Measure the signal value in the detection area.

[0027] In some examples, the first judgment condition includes: after a first time period or the amount of sample liquid is greater than or equal to a predetermined amount or the signal intensity of the signal molecules in the detection area is greater than or equal to a threshold.

[0028] In some examples, the first time period is in the range of 0 to 60 seconds, the predetermined amount is in the range of 15 μL to 50 μL, and the threshold is higher than the chip's reasonable background range. The range of signal intensity varies for different signal molecules and corresponding detection devices. The reasonable background range referred to herein is an intensity greater than the background signal, i.e., clearly indicating the presence of corresponding signal molecule intensities.

[0029] In some examples, the second time period is in the range of 10 seconds to 300 seconds, and the third time period is in the range of 30 seconds to 5 minutes.

[0030] The beneficial effects of the microfluidic chip and its detection method obtained through the above technical solution are:

[0031] 1. Because the sample loading area is closer to the detection area, the flow distance of the sample in the microchannel is reduced, which reduces the amount of sample used. Furthermore, the sample can be added directly to react with the detection area. After the sample reacts in the detection area, the buffer solution is used to push the signal molecules, avoiding the dilution of the sample caused by directly pushing the sample with the buffer solution. This results in higher sensitivity and more accurate detection results.

[0032] 2. It can directly detect whole blood or highly viscous samples, and has a wide range of sample applicability. The signal molecules in the labeled region are propelled into the reaction region by the buffer solution, rather than being directly propelled by the sample. This avoids contamination of the channel surface by whole blood or highly viscous samples, which could affect the accuracy of subsequent detections.

[0033] 3. Excess sample is absorbed by the absorbent material after the reaction in the reaction zone, avoiding the influence of other substances in the sample on the signal molecules.

[0034] 4. It can overcome the barbed effect of immune response, avoiding situations where the content of target analytes in some samples is too high (far exceeding the number of antigens / antibodies labeled by signal molecules), causing the target analytes to fill the reaction sites of the entire reaction area. As a result, the immune complexes formed by the target analytes and signal molecules cannot or rarely react with the antigens / antibodies in the detection area, making it impossible to accurately detect the signal value in the detection area and affecting the accuracy of the detection results.

[0035] In another aspect, the present invention proposes a microfluidic chip, comprising a substrate; a cover plate including a groove extending along its length; and a microchannel formed by the substrate and the groove, wherein the groove of the cover plate includes a channel region, and the channel region is sequentially provided with a sample application region, a reaction region, and a control region along its length, the sample application region, the reaction region, and the control region being in fluid communication with each other, wherein a control valve is provided in the control region, the control valve being configured to control the flow of liquid from the reaction region to the control region, and wherein the height of the reaction region is lower than the height of the control region, and the width of the reaction region is smaller than the width of the control region.

[0036] In some examples, the control region gradually narrows towards the reaction region.

[0037] In some examples, the control valve includes a movable fluid suction element and a braking port, the fluid suction element being able to contact or move away from the reaction zone, and the braking port being located on the side away from the reaction zone, wherein the braking port is a rectangular through hole penetrating the substrate and the cover plate, and the fluid suction element having a through hole at the end away from the reaction zone, the through hole being located within the braking port.

[0038] In some examples, the control area is provided with a locking part configured to hold the fluid suction element in a position away from the reaction area.

[0039] In some examples, the locking portion includes a protrusion arranged in the width direction, and the fluid suction member is correspondingly provided with a recess that mates with the protrusion.

[0040] In some examples, the cover has a retention portion at the junction of the control zone and the reaction zone, the retention portion extending at least partially into the reaction zone, such that when the fluid suction member comes into contact with a channel in the reaction zone, at least a portion of the fluid suction member is inserted into the retention portion.

[0041] In some examples, the retention portion is a toothed portion, which is constructed as a concave arc extending into the reaction zone. The lower end of the arc is provided with a serrated or wavy structure. The fluid suction member gradually narrows on the side near the reaction zone and has a notch in the middle on the side near the reaction zone, so that when the fluid suction member contacts the channel of the reaction zone, the notch is inserted into the toothed portion.

[0042] In some examples, the retention section is an inclined groove that slopes toward the reaction zone.

[0043] In some examples, the fluid suction device includes a water-absorbing material, wherein the water-absorbing material includes at least one of a desiccant, an antioxidant, and a moisture indicator.

[0044] In some examples, the sample application area is provided with a sample application hole that connects the upper and lower surfaces of the cover plate, and the height of the sample application hole on the lower surface of the cover plate is at the same horizontal plane as the height of the reaction area.

[0045] In some examples, the groove is rectangular, the shape of the substrate matches the groove, and the substrate is placed in the groove on the same horizontal plane as the surface of the cover plate.

[0046] In some examples, the groove of the cover sheet is provided with a channel area, a barrier area and a pressing area along the width direction from the middle to the outside. The height of the channel area and the barrier area is higher than the height of the pressing area, so that the substrate is placed in the groove on the lower surface of the cover sheet and contacts the pressing area, and a gap is formed between the substrate and the channel area and the barrier area.

[0047] In some examples, the barrier region is located around the sample application area and / or reaction area of ​​the channel area, and in the length direction, the barrier region covers at least a portion of the reaction area, or covers the sample application area and / or reaction area.

[0048] In some examples, the substrate is coated with a hydrophobic material at the location of the barrier area corresponding to the groove in the cover plate.

[0049] In some examples, the reaction zone and the barrier zone are on the same horizontal plane, and the barrier zone is also coated with a hydrophobic material.

[0050] In some examples, the substrate is laser-processed at the location of the barrier area corresponding to the groove in the cover plate to form a hydrophobic surface.

[0051] In some examples, the reaction region and the barrier region are on the same horizontal plane, and the barrier region is laser-treated to form a hydrophobic surface.

[0052] In some examples, the height of the reaction zone is lower than the height of the barrier zone, so that the sample will not enter the barrier zone due to gravity as it flows along the reaction zone.

[0053] The beneficial effects of the microfluidic chip obtained through the above technical solution are:

[0054] 1. A control valve is installed in the control zone. The control valve controls the flow of liquid from the reaction zone to the control zone, so that the liquid in the reaction zone flows to the control zone when needed, thereby controlling the time in the sample reaction zone, so that it can flow to the control zone after fully reacting, and not stagnate in the reaction zone channel.

[0055] 2. The teeth of the control valve allow the fluid suction device to be inserted into the control and reaction zone. The top of the device is inserted into the arc-shaped structure of the reaction zone, and the bottom contacts the serrated or wavy structure at the bottom of the arc-shaped structure. The fibers inside the fluid suction device come into contact with the serrated or wavy structure and then interlock, causing the bottom of the fluid suction device to be squeezed and thickened, so that it can better contact the channel of the reaction zone and achieve the purpose of adsorbing and retaining the sample in the channel.

[0056] 3. The control valve can be used multiple times in a single experiment to open and close the channel between the control reaction zone and the control zone, and the liquid in the channel can be adsorbed multiple times over an appropriate period of time, thereby increasing the accuracy of the detection.

[0057] 4. The substrate is placed in the corresponding barrier area of ​​the cover plate groove and coated with a hydrophobic material or laser treated so that the sample is restricted to flow in the corresponding reaction area during the sample addition process and will not flow into the corresponding barrier area.

[0058] 5. The barrier zone on the cover is higher than the reaction zone, so that the sample will not enter the barrier zone due to gravity when it flows along the reaction zone, thus achieving the purpose of the sample flowing in the reaction zone.

[0059] 6. When the barrier zone and the reaction zone are at the same height, the barrier zone on the cover is coated with a hydrophobic material or laser-treated to similarly restrict the flow of the sample within the reaction zone. Attached Figure Description

[0060] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this disclosure and are not intended to limit this disclosure.

[0061] Figure 1 is a top view of the microfluidic chip according to at least one embodiment of the present invention;

[0062] Figure 2 is a schematic diagram of the cover sheet structure of a microfluidic chip according to at least one embodiment of the present invention;

[0063] Figure 3 is a schematic diagram of the cross-sectional structure taken along line AA in Figure 2;

[0064] Figure 4 is an enlarged view of point B in Figure 2, which shows the retention part of the tooth;

[0065] Figure 5 is an enlarged view of point C in Figure 3, showing the cross-sectional structure of the retention section;

[0066] Figure 6 is a schematic diagram of the cover sheet structure of a microfluidic chip according to another embodiment of the present invention;

[0067] Figure 7 is a schematic diagram of the cross-sectional structure taken along line DD in Figure 6;

[0068] Figure 8 is an enlarged view of point E in Figure 6;

[0069] Figure 9 is an enlarged view of point F in Figure 7;

[0070] Figure 10 is a schematic diagram of the structure of a substrate according to at least one embodiment of the present invention;

[0071] Figure 11 is a side cross-sectional view of a microfluidic chip according to at least one embodiment of the present invention, showing the groove of the cover plate.

[0072] Figure 12 is a side cross-sectional view of a microfluidic chip according to another embodiment of the present invention; it shows the groove of the cover plate.

[0073] Figure 13 is a schematic diagram of the cover plate at the groove position according to another embodiment of the present invention;

[0074] Figure 14 is a side cross-sectional view of a microfluidic chip according to another embodiment of the present invention; it shows the groove of the cover plate;

[0075] Figure 15 is a schematic diagram of the structure of a microfluidic chip according to at least one embodiment of the present invention;

[0076] Figure 16 is a schematic cross-sectional view of a microfluidic chip according to at least one embodiment of the present invention;

[0077] Figure 17 is a schematic diagram of the structure of a microfluidic chip according to another embodiment of the present invention.

[0078] In Figures 1 to 14, 1-substrate; 2-cover plate; 21-groove; 211-channel area; 2111-sample loading area; 2112-reaction area; 2113-control area; 2112a-control valve; 2113a1-fluid suction element; 2113a2-toothed part; 2113a3-brake hole; 212-barrier area; 213-pressing area; 3-microchannel; 4-through hole; 5-locking part.

[0079] In Figures 15 to 17, 1-substrate; 2-cover sheet; 3-microchannel; 4-absorbent material; 31-buffer solution loading area; 32-reaction area; 33-control area; 321-labeling area; 322-detection area; 323-sample loading area. Detailed Implementation

[0080] To address the shortcomings of existing technologies, this invention proposes a novel microfluidic chip and its detection method. The invention will now be described in detail with reference to the accompanying drawings. It should be noted that in the drawings, the same reference numerals are assigned to components having substantially the same or similar structure and function, and repeated descriptions of them are omitted.

[0081] Compared to the embodiments shown in the accompanying drawings, feasible embodiments within the scope of protection of this invention may have fewer components, other components not shown in the drawings, different components, components arranged differently, or components with different connections, etc. Furthermore, without departing from the spirit of this invention, two or more components in the drawings may be implemented in a single component, or a single component shown in the drawings may be implemented as multiple separate components.

[0082] As shown in Figures 1 to 5, a microfluidic chip according to at least one embodiment of the present invention includes a substrate 1, a cover plate 2, and a microchannel formed by the substrate 1 and the cover plate 2. A groove 21 is provided in the middle of the lower surface of the cover plate 2, and the groove 21 extends along the length direction L.

[0083] It should be noted that the length direction L in the attached figure refers to the direction along the length of the microfluidic chip, which is exemplarily a horizontal direction to the right in the figure. The width direction W in the attached figure refers to the direction along the width of the microfluidic chip, which is exemplarily a vertical downward direction in the figure.

[0084] As shown in Figure 1, the groove 21 of the cover plate 2 is provided with a channel area 211, a barrier area 212 and a pressing area 213 from the middle to the outside along the width direction W. The height of the channel area 211 and the barrier area 212 is higher than the height of the pressing area 213, so that the substrate 1 is placed in the groove 21 on the lower surface of the cover plate 2 and contacts the pressing area 213, and a gap is formed between the substrate 1 and the channel area 211 and the barrier area 212.

[0085] As shown in Figure 2, the channel area 211 is sequentially arranged with a sample application area 2111, a reaction area 2112, and a control area 2113 along the liquid flow direction (length direction L). The sample application area 2111, reaction area 2112, and control area 2113 are connected. The height of the reaction area 2112 is lower than the height of the control area 2113, and the width of the control area 2113 is greater than the width of the reaction area 2112. Considering the characteristics of the control valve 2113a and the retention section 2113a2 described later, the height difference between the reaction area 2112 and the control area 2113 helps to stably maintain the fluid suction element 2113a1 in contact with the reaction area 2112. Furthermore, the smaller width of the reaction area 2112 compared to the control area 2113 helps to absorb excess liquid into and contain it within the control area 2113.

[0086] For example, the control zone 2113 can gradually narrow from the middle to the reaction zone 2112.

[0087] In this embodiment, a control valve 2113a is provided in the control zone 2113. The control valve 2113a is configured to control the flow of liquid from the reaction zone 2112 to the control zone 2113, so that the liquid in the reaction zone 2112 flows to the control zone when needed, thereby controlling the time in the sample reaction zone 2112, so that it can fully react and then flow to the control zone 2113, and not stagnate in the channel of the reaction zone 2112.

[0088] Specifically, the control valve 2113a can be implemented in various ways. For example, in one embodiment shown in Figures 2 to 5, the control valve 2113a may include a brake port 2113a3 and a movable fluid suction member 2113a1, which can contact or move away from the reaction zone 2112. When the fluid suction member 2113a1 contacts the reaction zone 2112, it can draw liquid from the reaction zone 2112 into the control zone 2113. Conversely, when the fluid suction member 2113a1 moves away from or does not contact the reaction zone 2112, the liquid remains in the reaction zone 2112 and does not flow into the control zone 2113.

[0089] Referring again to Figure 2, to prevent the fluid suction member 2113a1 from accidentally contacting the reaction zone 2112, the control zone 2113 is provided with a locking part 5, which is configured to hold the fluid suction member 2113a1 away from the reaction zone 2112. In this embodiment, the locking part 5 includes a protrusion arranged along the width direction W, and the fluid suction member 2113a1 is correspondingly provided with a recess that mates with the protrusion. The protrusion can be arc-shaped or rectangular, and can be arranged at the same position or staggered in the length direction.

[0090] Alternatively, the locking part 5 can be other possible stopping structures, such as a snap-fit ​​structure or an elastic reset member that biases the fluid suction member 2113a1 toward the reaction zone 2112.

[0091] The cover plate 2 is provided with a retention part 2113a2 at the connection between the control zone 2113 and the reaction zone 2112. The retention part 2113a2 extends at least partially into the reaction zone 2112, so that when the fluid suction member 2113a1 comes into contact with the channel of the reaction zone 2112, at least a part of the fluid suction member 2113a1 is inserted into the retention part 2113a2.

[0092] As shown in Figures 4 and 5, the retention portion 2113a2 can be toothed, and its structure is a concave arc extending into the reaction zone 2112, with serrations at the lower end of the arc. The fluid suction member 2113a1 gradually narrows on the side near the reaction zone 2112, and a notch is provided in the middle of the side near the reaction zone 2112, so that when the fluid suction member 2113a1 contacts the channel of the reaction zone 2112, the notch inserts into the toothed portion.

[0093] Alternatively, in embodiments not shown, the lower end of the arc can also be configured as a wave structure. Unlike sawtooth patterns, a wave structure can be an undulating curve.

[0094] Therefore, when the fluid suction element 2113a1 is inserted into the control zone 2113 and contacts the reaction zone 2112, the top is inserted into the arc-shaped structure of the reaction zone 2112, and the bottom contacts the serrations at the bottom of the arc-shaped structure. The fibers inside the fluid suction element 2113a1 come into contact with the serrations and then interlock, causing the bottom of the fluid suction element 2113a1 to be squeezed and thickened, so that it can better contact the channel of the reaction zone 2112 and achieve the purpose of adsorbing and retaining the sample in the channel.

[0095] The movement of the fluid suction member 2113a1 can be achieved through the structure of the braking hole 2113a3 and the through hole 4. As shown in Figure 2, the braking hole 2113a3 is a rectangular through hole penetrating the substrate cover. The end of the fluid suction member 2113a1 away from the reaction zone can be provided with the through hole 4, which is located within the range of the braking hole 2113a3. For example, a brake rod can be inserted into the braking hole 2113a3 and pass through the through hole 4 on the fluid suction member 2113a1 to move the fluid suction member 2113a1, so that the other end of the fluid suction member 2113a1 contacts or leaves the reaction zone 2112. The liquid in the reaction zone 2112 can be controlled to be adsorbed onto the fluid suction member 2113a1 at a suitable time.

[0096] For example, Figures 6 to 9 also illustrate a cover structure for a microfluidic chip according to another embodiment of the present invention. It should be noted that the following description of the embodiment mainly focuses on its differences from the aforementioned embodiments; identical or similar features will be omitted. Unlike the aforementioned embodiments, this embodiment employs a simpler retention portion and a different type of locking portion. As shown in Figures 6 to 9, the retention portion 2113a2 is an inclined groove oriented towards the reaction zone 2112. The inclined groove can also accommodate and crosslink the fiber structure of the fluid suction member 2113a1. Even if its effect is not as good as the toothed portion, its processing is simpler and the cost is lower.

[0097] Furthermore, in this embodiment, the protrusion of the locking portion 5 is rectangular, and the recess of the fluid suction member 2113a1 is a recess that mates with the rectangular protrusion. Since the rectangular width of the recess is relatively small, it is shown as a straight line in FIG6. This is because the width of the rectangular recess should not be too large, otherwise it will affect the movement of the fluid suction member 2113a1.

[0098] Alternatively, the control valve 2113a can also use structures known in the art, such as pneumatic valves or plunger pumps, to control the flow of liquid, which will not be described in detail here.

[0099] For example, the fluid suction element 2113a1 is made of a water-absorbing material, which may include or incorporate one or more of a desiccant, an antioxidant, and a moisture indicator.

[0100] For example, absorbent materials can be polyester fibers, absorbent resins, absorbent gelatin, paper pulp, or other materials with absorbent properties.

[0101] For example, the reaction zone 2112 is provided with a labeling zone, a detection zone, and a quality control zone along the length direction L, which can also be called the sample flow direction.

[0102] The groove 21 in the middle of the cover plate 2 is rectangular. The shape of the substrate 1 matches the groove 21. The substrate 1 is placed in the groove 21 and is on the same horizontal plane as the surface of the cover plate 2.

[0103] The sample application area 2111 is provided with a sample application hole that connects the upper and lower surfaces of the cover plate 2. The height of the sample application hole on the lower surface of the cover plate 2 is at the same level as the height of the reaction area 2112, so that the sample flows through the sample application hole to the reaction area 2112 on the right.

[0104] The barrier region 212 is located around the reaction region 2112 in the channel region 211, and in the length direction L, the barrier region 212 covers the entire reaction region 2112. As shown in Figure 10, the substrate 1 can be coated with a hydrophobic material at the position of the barrier region 212 in the groove 21 of the cover plate 2, so that during the sample addition process, the bottom is restricted to flow within the reaction region 2112 and will not flow into the barrier region 212.

[0105] Alternatively, in embodiments not shown, the barrier region 212 may only cover part of the reaction region 2112, or it may only be provided in the sample application region 2111 and cover the sample application region 2111, or it may be provided around the sample application region 2111 and the reaction region 2112 and cover the sample application region 2111 and the reaction region 2112.

[0106] As shown in Figures 13 and 14, the reaction zone 2112 and the barrier zone 212 are on the same horizontal plane. The barrier zone 212 is also coated with a hydrophobic material to confine the sample flow within the reaction zone 2112. By coating the barrier zone 212 with a hydrophobic material, during the reaction, it is ensured that the sample liquid is confined within the channels of the reaction zone 2112 and does not leak or flow into non-reaction zones, such as the barrier zone 212, thereby improving the reaction sensitivity.

[0107] For example, the hydrophobic material may be polytetrafluoroethylene ink, silane coupling agent ink, fluoride polymer ink, or other materials with hydrophobic properties.

[0108] As shown in Figure 11, the height of the reaction zone 2112 is lower than the height of the barrier zone 212, so that when the sample flows along the reaction zone 2112, it will not enter the barrier zone 212 due to gravity. Therefore, it is not necessary to add hydrophobic materials to the barrier zone 212, and the flow of the sample in the reaction zone 2112 can also be restricted.

[0109] It should be noted that the solid-color filled areas in Figures 11 and 13 represent the coated hydrophobic material, which is shown as thicker only for ease of description. In reality, the coated hydrophobic material can be a very thin layer.

[0110] Alternatively, at the locations where hydrophobic materials are coated in Figures 10 and 11, the existing hydrophilic layer can be removed using laser processing to form a hydrophobic surface, as shown in Figure 12. Laser processing can be, for example, laser sintering or laser engraving. The location and size of the laser processing can be consistent with the locations where hydrophobic materials are coated in the aforementioned embodiments.

[0111] After laser treatment, the original hydrophilic layer is removed, and a concave structure is formed. This concave structure itself is hydrophobic. In addition, due to the high temperature of the laser, the side of the concave structure near the channel is dissolved and forms a protrusion, further creating a physical barrier that prevents liquid from flowing into the concave structure.

[0112] Alternatively, in embodiments where the reaction region 2112 and the barrier region 212 are on the same horizontal plane, the barrier region 212 can also be directly laser-treated to form a hydrophobic surface. In this case, the substrate 1 can either be coated with a hydrophobic material at the location corresponding to the barrier region 212, or it can be laser-treated as shown in FIG12.

[0113] The following are experimental examples of several embodiments of the microfluidic chip of the present invention to verify and illustrate the technical effects claimed by the present invention.

[0114] Example 1

[0115] Experimental objective: To compare the impact of adding a control valve to the waste liquid area (control area) on the interpretation results.

[0116] 1. Material preparation

[0117] The improved microfluidic chip No. 1 (before improvement) and the improved microfluidic chip No. 2 (with a control valve) are compared. The improved chip No. 2, compared to the original chip No. 1, has a control valve added to the waste liquid zone (control zone) at the end of its reaction zone. All of the above were manufactured by Shandong Mai Microbial Technology Co., Ltd.

[0118] RSV clinical samples were obtained from the relevant hospitals;

[0119] Shandong Mai Microbial Technology Co., Ltd. produces fluorescence immunoassay analyzers, timers (such as stopwatches), and pipettes.

[0120] 2. Encapsulation site

[0121] The coating spot is located in the detection area and is coated with RSV antibodies;

[0122] The second coating point is located in the quality control area and is coated with a secondary antibody.

[0123] The fluorescently labeled region is immobilized with dried fluorescently labeled paired antibodies.

[0124] 3. Detection Method

[0125] 3.1 Improved microfluidic chip with control valve (chip 2)

[0126] The improved microfluidic chip with a control valve (chip 2) was placed flat on the experimental stage. RSV standard solution samples with concentrations of 100 ng / ml, 5 ng / ml, and 1 ng / ml were added to the sample wells of the microfluidic chip. After 90 seconds, the control valve was used to allow the absorbent material to contact the reaction zone, causing the liquid in the reaction zone to flow to the control zone. The chip was then read using a fluorescence immunoassay analyzer, and the detection results of the IgE samples were recorded. Each sample was tested twice, and the detection signal value, the control zone (C line) signal value, and the detection zone (T line) signal value were recorded for each sample.

[0127] 3.2 Microfluidic chip before improvement (chip 1)

[0128] The microfluidic chip (chip 1) before the improvement was placed flat on the experimental stage. RSV standard solution samples with concentrations of 100 ng / ml, 5 ng / ml, and 1 ng / ml were added to the sample wells of the microfluidic chip. After the samples reacted, they flowed naturally to the waste liquid area at the end of the reaction zone and were adsorbed by the water-absorbing material. The chip was read using a fluorescence immunoassay analyzer, and the detection results of the IgE samples were recorded. Each sample was tested twice, and the detection signal value, control zone (C line), and detection zone (T line) signal values ​​of each sample were recorded.

[0129] 4. Results

[0130] As shown in Table 1, the improved microfluidic chip No. 2 with a control valve uses the control valve to control the reaction and then connect the reaction zone and the control zone after 90 seconds, so that the sample can fully react in the reaction zone. The detected T-line and C-line signal values ​​are higher than those of the original microfluidic chip No. 1.

[0131] Table 1. Sample detection results before and after the microfluidic chip improvement

[0132] Experimental conclusion: Adding a control valve to the waste liquid zone (control zone) at the end of the reaction zone controls the flow of liquid from the reaction zone to the control zone, thereby controlling the time in the sample reaction zone. This allows the liquid in the reaction zone to flow to the control zone after sufficient reaction, resulting in higher detected signal values ​​and greater accuracy.

[0133] Example 2

[0134] Experimental objective: To compare the water absorption rate after adding a retention section at the end of the reaction zone and its impact on the interpretation results.

[0135] 1. Material preparation

[0136] The improved IgE3 microfluidic chip (before improvement) and the improved toothed IgE4 microfluidic chip (after improvement) have a retention section (toothed section in this example) added to the top cover of the channel at the end of the reaction zone (near the absorbent material end). All of the above were manufactured by Shandong Mai Microbial Technology Co., Ltd.

[0137] IgE clinical serum sample S1 was obtained from the relevant hospital;

[0138] Shandong Mai Microbial Technology Co., Ltd. produces fluorescence immunoassay analyzers, timers (such as stopwatches), and pipettes.

[0139] 2. Encapsulation site

[0140] The coating spot is located in the detection area and is coated with IgE antibodies;

[0141] The second coating point is located in the quality control area and is coated with a secondary antibody.

[0142] The fluorescently labeled region is immobilized with dried IgE fluorescently labeled paired antibody.

[0143] 3. Detection Method

[0144] 3.1 Improved Tooth IgE Microfluidic Chip

[0145] The improved toothed microfluidic chip (chip 4) was placed flat on the experimental stage. 35 μL of sample was added to the sample well of the microfluidic chip. After 90 seconds, the absorbent material was brought into contact with the channel to absorb water. The time it took for the absorbent material to absorb the liquid until the flow stopped was recorded. The chip was then analyzed using a fluorescence immunoassay analyzer, and the detection results of the IgE samples were recorded. Each sample was tested three times, and the detection signal value, the peak value of the control zone (C line), and the peak value of the detection zone (T line) were recorded for each sample.

[0146] 3.2 IgE microfluidic chip before improvement

[0147] Before the improvement, the IgE microfluidic chip (chip 3) was placed flat on the experimental stage. 35 μL of sample was added to the sample well of the microfluidic chip, and after 90 seconds, the absorbent material was brought into contact with the channel to absorb water. The time it took for the absorbent material to absorb the liquid until the flow stopped was recorded. The chip was then interpreted using a fluorescence immunoassay analyzer, and the detection results of the IgE sample were recorded. Each sample was tested three times, and the detection signal value, the peak value of the control zone (C line), and the peak value of the detection zone (T line) were recorded for each sample.

[0148] 4. Results

[0149] As shown in Table 2, the water absorption time of chip #4 is within 2 minutes and 30 seconds, while that of chip #3 is approximately 3 minutes. There is no significant difference in signal values. The improved water absorption time is 30 seconds shorter than the original chip.

[0150] Table 2 Sample detection results before and after microfluidic chip improvement

[0151] Experimental conclusion: Adding teeth to the end of the reaction zone has a guiding effect on the liquid, increasing the contact area between the liquid and the absorbent material, resulting in a faster water absorption rate than the original structure, with almost no impact on the interpretation results. This can optimize the instrument's interpretation speed.

[0152] Example 3

[0153] Experimental objective: To compare and verify the effect of coating both sides of the corresponding upper cover channel position on the interpretation results.

[0154] 1. Material preparation

[0155] The improved version of the previous quadruple assay (RSV / FluA / FluB / COVID-19) microfluidic chip No. 5 and the quadruple assay (test items same as above) microfluidic chip No. 6, coated with hydrophobic material, differs from chip No. 5 in that the substrate of chip No. 6 is sprayed with hydrophobic coating at the corresponding channel positions after assembly with the top cover. This prevents sample leakage from the channels or sides of the reaction area due to excessive sample addition or excessively high microsphere concentration, which would affect the test results. Both were manufactured by Shandong Mai Microbial Technology Co., Ltd.

[0156] RSV clinical sample S2 was obtained from the relevant hospital;

[0157] Shandong Mai Microbial Technology Co., Ltd. produces fluorescence immunoassay analyzers, timers (such as stopwatches), and pipettes.

[0158] 2. Encapsulation site

[0159] The coating spot is located in the detection area and is coated with RSV antibodies;

[0160] The second coating spot is located in the detection area and is coated with FluA antibody.

[0161] The coating spot 3 is located in the detection area and is coated with FluB antibody;

[0162] Spot 4, located in the testing area, is coated with COVID-19 antibodies;

[0163] The coating point 5 is located in the quality control area, and the coating contains secondary antibodies.

[0164] The fluorescently labeled region is immobilized with dried fluorescently labeled paired antibodies.

[0165] 3. Detection Method

[0166] 3.1 Quadruple microfluidic chip coated with hydrophobic material

[0167] A quadruple microfluidic chip (chip 6) coated with hydrophobic material was placed flat on the experimental stage. A 35 μL RSV sample was added to the sample well. After timing for 90 seconds, the absorbent material was allowed to absorb water for 3 minutes using a control valve. The chip was then read using a fluorescence immunoassay analyzer, and the detection results of the RSV sample were recorded. Each sample was tested three times, and any leakage was observed after sample addition. The detection signal value, peak value of the control zone (C line), and peak value of the detection zone (T line) for each sample were recorded.

[0168] 3.2 Improved Quadruple Microfluidic Chip

[0169] Before the improvement, the four-cell microfluidic chip (chip 5) was placed flat on the experimental stage. 35 μL of RSV sample was added to the sample well of the microfluidic chip. After timing for 90 seconds, the absorbent material was allowed to absorb water for 3 minutes using a control valve. The chip was then read using a fluorescence immunoassay analyzer, and the detection results of the RSV sample were recorded. Each sample was tested three times, and any leakage was observed after sample addition. The detection signal value, peak value of the control zone (C line), and peak value of the detection zone (T line) for each sample were recorded.

[0170] 4. Results

[0171] As shown in Table 3, even if leakage occurs in the No. 6 quadruple-sensor microfluidic chip coated with hydrophobic material, the hydrophobic material can intercept it, preventing microspheres from scattering around the channel and affecting the detection background, and also preventing liquid leakage in the reaction zone from affecting the flow of microspheres into the channel. When the hydrophilic material absorbs water, it can absorb the intercepted liquid without affecting the detection results. The previous No. 5 quadruple-sensor microfluidic chip also experienced leakage, with microsphere leakage in the reaction zone rendering the results invalid. Although the hydrophilic material can also absorb the leaked liquid, the non-specific adsorption of microspheres in incorrect locations can still affect the interpretation results.

[0172] Table 3 Sample detection results before and after microfluidic chip improvement

[0173] Experimental conclusion: Leakage has a certain probability of occurring, but coating with hydrophobic materials can effectively intercept leaked liquid and ensure the accuracy of the readings.

[0174] Example 4

[0175] Experimental objective: To verify the advantages of the control region of the dual-drive microfluidic chip in detecting IgE in plasma samples by repeatedly (more than two times) contacting / disconnecting from the channel.

[0176] 1. Material preparation

[0177] The improved IgE microfluidic chips No. 7 and No. 8 were used for detection, employing a one-step method (chip No. 7): one contact with the control area through the channel, and a two-step method (chip No. 8): two contact with the control area through the channel. Both were manufactured by Shandong Mai Microbial Technology Co., Ltd.

[0178] IgE clinical plasma sample S3 was obtained from the relevant hospital;

[0179] Shandong Mai Microbial Technology Co., Ltd. produces fluorescence immunoassay analyzers, timers (such as stopwatches), and pipettes.

[0180] 2. Encapsulation site

[0181] The coating spot is located in the detection area and is coated with anti-human IgE antibodies;

[0182] The second coating point is located in the quality control area and is coated with biotinylated IgE antigen.

[0183] The labeled region is fixed with dried IgE fluorescently labeled paired antibodies.

[0184] 3. Detection Method

[0185] 3.1 The dual-drive microfluidic chip controls the filter in the control zone to twice disconnect from / contact the channel to control the reaction process.

[0186] Place the IgE microfluidic chip (chip 8) flat on the experimental stage. Add 20 μL of IgE plasma sample to the sample well. After 10 seconds, the plasma flows through the detection zone and control zone to the top of the channel. At this point, open the control valve to allow the absorbent material to contact the channel, drawing away excess plasma until the flow stops (first contact). Allow the plasma sample remaining in the channel to react with the antibody in the detection zone for 90 seconds. Then, add 35 μL of sheep serum as a buffer solution to the sample well. After the buffer solution merges with the plasma in the channel, continue flowing towards the control zone for about 10 seconds. Once the plasma in the channel is drawn away and the fluorescent microspheres dissolve and fill the channel, open the control valve to disconnect the absorbent material from the channel (absorbent material detachment). After 2 minutes, use the absorbent material again to absorb water for 2 minutes (second contact). Then, use a fluorescence immunoassay analyzer to interpret the chip and record the detection results of the IgE sample. Each sample is tested three times. Record the peak value of the control zone (C line) and the peak value of the detection zone (T line) for each sample.

[0187] 3.2 Dual-drive microfluidic chip control zone filter single-stage detachment / contact channel to control reaction process

[0188] The IgE microfluidic chip (chip 7) was placed flat on the experimental stage. 20 μL of plasma sample was added to the micro-sample well. After 10 seconds, the sample flowed through the detection and control zones. At this point, the control valve caused the absorbent material to contact the channel, drawing away excess plasma until the flow stopped (first contact). The plasma sample remaining in the channel reacted with the antibody for 90 seconds. 35 μL of sheep serum was added to the sample well as a buffer solution. After the fluorescent microspheres dissolved, they flowed through the channel and continued into the absorbent material in the control zone. After 2 minutes of absorption, the chip was read using a fluorescence immunoassay analyzer. The IgE sample detection results were recorded. Each sample was tested three times, and the peak values ​​of the control zone (C line) and detection zone (T line) were recorded for each sample.

[0189] 4. Results

[0190] As shown in Table 4, the peak values ​​of the C-line and T-line of IgE at 6 min were relatively consistent in the two-step controlled microfluidic chip (chip 8), while the peak value of the C-line of IgE detected by the one-step controlled microfluidic chip (chip 7) was lower (possibly due to the remaining IgE competing with the IgE antigen in the C region for binding to the antibody on the fluorescent microspheres), and the T-line value was higher in the two-step method than in the one-step method.

[0191] Table 4. Sample detection results before and after the microfluidic chip improvement

[0192] Experimental conclusion: The detection results of the two-step contact / disengagement control reaction are significantly better than those of the one-step control reaction.

[0193] Example 5

[0194] Experimental objective: To compare and verify the effects of coating the substrate with hydrophobic material on both sides of the corresponding channel position and laser treatment on the interpretation results.

[0195] 1. Material preparation

[0196] The four-piece test (test items as above) of microfluidic chip No. 9 coated with hydrophobic material and microfluidic chip No. 10 after laser treatment. The difference between chip No. 10 and chip No. 9 is whether chip No. 9 is coated with hydrophobic material or laser-sintered. Both were manufactured by Shandong Mai Microbial Technology Co., Ltd.

[0197] RSV clinical sample S2 was obtained from the relevant hospital.

[0198] 2. Detection Method

[0199] 2.1 Quadruple microfluidic chip coated with hydrophobic material

[0200] A quadruple microfluidic chip (chip 9) coated with hydrophobic material was placed flat on the experimental stage. A 35 μL LSV sample was added to the sample well, and the flow time of the sample in the microchannel was recorded. Then, the time it took for the control valve to allow the absorbent material to absorb water was recorded, and the sample was allowed to stand for 90 minutes. Each sample was tested three times to observe whether there was any leakage after sample addition.

[0201] 3.2 Laser-processed quadruple microfluidic chip

[0202] The laser-treated quadruple microfluidic chip (chip 10) was placed flat on the experimental stage. A 35 μL LSV sample was added to the sample well, and the flow time of the sample in the microchannel was recorded. Then, the time it took for the control valve to allow the absorbent material to absorb water was recorded, and the sample was allowed to stand for 90 minutes. Each sample was tested three times to observe whether there was any leakage after sample addition.

[0203] 4. Results

[0204] As shown in Table 5, the liquid retention capacity of the Quad-Test No. 9 microfluidic chip coated with hydrophobic material and the Quad-Test No. 10 microfluidic chip treated with laser were the same, and no leakage was observed. The laser-treated No. 10 chip did not affect the flow of liquid in the channel and had a shorter required water absorption time.

[0205] Table 5. Sample detection results before and after the microfluidic chip improvement

[0206] Experimental conclusions: The liquid retention capacity of the laser-treated hydrophobic surface is consistent with that of the chip coated with hydrophobic material. No leakage was observed after 90 minutes of standing, and the liquid flow in the channels was not affected; all processes were completed in approximately 15 seconds. Furthermore, the laser-etched hydrophobic surface showed a significant reduction in subsequent water absorption time.

[0207] In known microfluidic chips, because the sample loading port is located upstream of the labeling region, the sample will first react in the labeling region after passing through the loading port, and then enter the detection region. For highly viscous samples, they cannot push the signal molecules in the labeling region to the detection region by themselves, so a subsequent buffer solution is required to push them. However, this method will dilute the sample, causing inaccurate detection results. At the same time, other substances contained in the sample may also interfere with the signal molecules in the labeling region.

[0208] Furthermore, the hook effect of the immune response is also a problem that urgently needs to be addressed. The hook effect refers to the phenomenon of false negatives caused by an inappropriate antigen-antibody ratio. The detection signal intensity decreases as the antigen concentration increases. This is because the target analyte occupies the entire reaction site of the reaction zone, while the immune complex formed by the target analyte and the signal molecule cannot or minimally react with the antigen / antibody in the detection zone, making it impossible to accurately detect the signal value in the detection zone.

[0209] In another aspect, addressing the shortcomings of the prior art, this invention proposes a novel microfluidic chip and its detection method. The invention will now be described in detail with reference to the accompanying drawings. It should be noted that in the drawings, the same reference numerals are assigned to components having substantially the same or similar structure and function, and repeated descriptions of them are omitted.

[0210] As shown in Figures 15 and 16, a microfluidic chip according to at least one embodiment of the present invention includes a substrate 1, a cover plate 2, and a microchannel 3 formed by the substrate 1 and the cover plate 2. A groove on the cover plate 2 along its length direction L surrounds the substrate 1 to form the microchannel. The microchannel 3 includes a buffer solution loading area 31, a reaction area 32, and a control area 33. The buffer solution loading area 31, the reaction area 32, and the control area 33 are sequentially connected from left to right along the length direction L. A movable absorbent material 4 is disposed within the control area 33. The absorbent material 4 can be moved to contact or move away from the reaction zone 32. It can move to contact the reaction zone 32 as needed within a suitable time to adsorb excess sample in the microchannel 3. The reaction zone 32 includes a labeling zone 321, a detection zone 322, and a sample loading zone 323. The detection zone 322 and the sample loading zone 323 are both located on the same side of the labeling zone 321 (as shown in Figure 15, to the right of the labeling zone 321). After the sample enters the microchannel through the sample loading zone 33, it first flows into the detection zone 32.

[0211] It should be noted that the length direction L in the attached figure refers to the direction along the length of the microfluidic chip, which is exemplarily the horizontal rightward direction in the figure.

[0212] Specifically, detection sites and quality control sites are arranged sequentially from left to right along the length direction L on the detection area 322. The number of detection sites and quality control sites is one or more. The detection sites are used to detect the presence or concentration of the target analyte, and the quality control sites are used to verify the effectiveness of the reaction.

[0213] Specifically, the labeling region 321 includes an antibody / antigen labeled with a signal molecule, the detection site of the detection region 322 is coated with an antibody / antigen capable of binding to the target analyte, the target analyte can simultaneously bind to the labeling region 321 and the antibody / antigen at the detection site to form a double antigen / antibody sandwich complex, and the quality control site of the detection region 322 is coated with an antibody / antigen capable of binding to the antibody / antigen labeled with the signal molecule in the labeling region.

[0214] A braking hole 331 is provided on the right side of the control area 33, which can at least partially extend to contact the absorbent material 4. The braking hole 331 is a rectangular through hole penetrating the substrate 1 and the cover plate 2. The through hole of the absorbent material 4 at the end away from the reaction area 32 is disposed in the braking hole 331 to allow control of the movement of the absorbent material 4. For example, a braking mechanism can be inserted into the braking hole 331 and pass through the through hole on the absorbent material 4, thereby controlling the movement of the absorbent material 4 by controlling the movement of the braking mechanism or controlling the movement of the microfluidic chip.

[0215] The absorbent material 4 can be polyester fiber, absorbent resin, absorbent gelatin, paper pulp, or other materials with absorbent properties.

[0216] The buffer solution loading area 31 is provided with a buffer solution well connecting the upper and lower surfaces of the cover plate 2, and the sample loading area 323 is provided with a sample loading well connecting the upper and lower surfaces of the cover plate 2. Buffer solution and sample can be added into the microchannel 3 through the buffer solution well and the sample loading well, respectively.

[0217] The sample loading area 323 and the marking area 321 are respectively located on both sides of the detection area 322. Therefore, after the sample enters the microchannel 3 through the sample loading area 323 or the loading hole, as shown in Figure 15, it flows to the left to the detection area 322 due to capillary action and combines with the detection area 322. Excess sample can be adsorbed by the water-absorbing material 4.

[0218] Alternatively, as shown in Figure 17, the sample loading area 323 can also be located between the labeling area and the detection area 322. After the sample enters the microchannel 3 through the sample loading area 323 or the loading hole, it moves to the left and right ends of the microchannel 3 due to capillary action. When it flows to the right, it flows into the detection area 322. After the reaction is completed, the excess sample is adsorbed by the water-absorbing material 4. However, this structure requires control of the amount of sample added so that the sample just binds to the detection area 322 and not to the labeling area 321.

[0219] For example, the antigen / antibody labeled with the signaling molecule can be an antigen / antibody labeled with the signaling molecule. The signaling molecule can be, for example, a fluorescent protein, a fluorescent dye, a fluorescent microsphere, or a nanoparticle, such as a quantum dot.

[0220] On the other hand, the present invention also provides a method for detecting microfluidic chips, using the microfluidic chip as described above for detection, the method comprising at least the following steps:

[0221] 1) Add the sample into the microchannel 3 through the sample addition area 323, and keep the absorbent material 4 away from the reaction area 32;

[0222] 2) Wait for the first time period. After the sample flows back through the detection zone 322 under capillary action, move the absorbent material 4 to contact the reaction zone 32 to absorb the excess sample until the flow stops, and let the sample remaining in the microchannel 3 react with the detection zone 322.

[0223] 3) After the second reaction time period, the buffer solution is added into the microchannel 3 through the buffer solution sample addition area 31. The buffer solution passes through the labeling area 321 under capillary action, and the antigen / antibody coated with the signal molecules in the labeling area 321 is washed away and enters the detection area 322. When the signal molecules in the channel of the reaction area 32 merge with the sample, the absorbent material 4 in the moving control area 33 contacts the reaction area 32, and the excess sample is adsorbed into the control area 33 through the absorbent material 4.

[0224] 4) After the excess sample in the channel is absorbed by the absorbent material 4 and the signal molecules in the labeled area are dissolved and spread throughout the reaction zone 32 channel, move the absorbent material 4 away from the reaction zone 32 so that the absorbent material 4 breaks the contact with the reaction zone 32 channel.

[0225] 5) After the third reaction time period, the water-absorbing material 4 in the moving control area 33 comes into contact with the detection area 322 and adsorbs the excess liquid, including signal molecules, into the control area 33 through the water-absorbing material 4.

[0226] 6) Finally, measure the signal value of the detection area 322. For example, a detector can be used to measure the signal value of the detection area for subsequent analysis (the detector can be a conventional technology, such as a fluorescence detector, which can be consistent with the signal carried by the antigen / antibody coated on the signal molecule).

[0227] For example, the first, second, and third time periods can be adjusted according to needs and sample conditions. For instance, the first time period can be in the range of 0 to 60 seconds, the second time period can be in the range of 10 to 300 seconds, and the third time period can be in the range of 30 seconds to 5 minutes.

[0228] The detection method can also determine when to move the absorbent material 4 to contact the reaction zone 32 to absorb excess sample until the flow is interrupted, based on a first judgment condition. The example above shows the process after a first time period. Alternatively, the first judgment condition may also include a sample liquid volume greater than or equal to a predetermined amount, wherein the predetermined amount is in the range of 15 μL to 50 μL; or a signal intensity of the signal molecules in the detection zone 322 greater than or equal to a threshold above the reasonable background range of the chip.

[0229] The following are experimental examples of several applications of the microfluidic chip of the present invention to verify and illustrate the technical effects claimed by the present invention.

[0230] It should be noted that the detection method proposed in this invention differs from the traditional microfluidic chip addition method (i.e., simultaneous addition of sample and buffer solution, also known as the "one-step method") because the chip used has both the detection area and the sample addition area located on the same side of the labeling area. The detection method proposed in this invention requires the separate addition of sample and buffer solution, and the sample needs to flow into the detection area after addition; therefore, it can also be called the "two-step method." In the embodiments described below, "one-step method" refers to the traditional microfluidic chip (also known as the chip before improvement), and "two-step method" refers to the microfluidic chip proposed in this invention (also known as the chip after improvement).

[0231] Example 6

[0232] Experimental objective: To verify whether one-step and two-step microfluidic chips can use whole blood as a test sample.

[0233] 1. Material preparation

[0234] The improved IgE microfluidic chip 1 (before improvement) and the improved two-step IgE microfluidic chip 2 (after improvement) differ in that the improved two-step microfluidic chip has an additional micro-sample dispensing port added to the cover plate on the reaction zone channel 0.5 cm to the left of the control area. Both were manufactured by Shandong Mai Microbial Technology Co., Ltd.

[0235] IgE clinical whole blood sample S2 was obtained from the relevant hospital;

[0236] Shandong Mai Microbial Technology Co., Ltd. produces fluorescence immunoassay analyzers, timers (such as stopwatches), and pipettes.

[0237] 2. Encapsulation site

[0238] The first coating point is located at detection site 322 in the detection area and is coated with IgE antibody.

[0239] The second coating site is located at the 322 quality control site in the detection area and is coated with a secondary antibody.

[0240] Labeled region 321 is fixed with dried IgE fluorescently labeled paired antibody.

[0241] 3. Detection Method

[0242] 3.1 Improved IgE Two-Step Microfluidic Chip

[0243] The improved IgE two-step microfluidic chip was placed flat on the experimental stage. 10 μL of whole blood sample was added to the micro-sample wells and allowed to flow backwards through the control and detection sites for 10 seconds. At this point, the absorbent material 4 was brought into contact with the channel to absorb excess whole blood until the flow stopped, allowing the remaining whole blood sample to react with the antibody for 90 seconds. 35 μL of sheep serum was added to the buffer well as a buffer solution. After the buffer solution and whole blood in the channel were combined, the absorbent material 4 was used to absorb the water for 10 seconds. Once the blood in the channel was absorbed and the signal molecules dissolved and filled the channel, the contact between the absorbent material 4 and the channel was disconnected. After reacting for 2 minutes, the absorbent material 4 was used again to absorb water for 2 minutes. The chip was then read using a fluorescence immunoassay analyzer, and the IgE sample detection results were recorded. Each sample was tested three times, and the signal values ​​at the control and detection sites were recorded for each sample.

[0244] 3.2 IgE microfluidic chip before improvement

[0245] Before the improvement, the IgE microfluidic chip was placed flat on the experimental stage. A 35 μL whole blood sample was added to the sample well of the microfluidic chip and reacted for 2 minutes. Then, absorbent material 4 was used to absorb water for 3 minutes. The chip was then read using a fluorescence immunoassay analyzer, and the detection results of the IgE sample were recorded. Each sample was tested three times, and the signal values ​​of the instrument's quality control site and detection site for each sample were recorded.

[0246] 4. Results

[0247] As shown in Table 6, the detection signal value, quality control site peak value, and detection site peak value of IgE in the two-step microfluidic chip 2 are relatively consistent at 6 minutes. In contrast, the IgE of the one-step microfluidic chip 1 is invalid within 5 minutes due to the significant background interference from whole blood, resulting in the inability to detect the quality control site peak value. Furthermore, the detection site value is higher in the two-step method than in the one-step method.

[0248] Table 6. Sample detection results before and after the microfluidic chip improvement

[0249] Experimental conclusion: The two-step method yields significantly better results than the one-step method. The one-step method, involving the addition of whole blood, leads to hemolysis, causing reddish background contamination and rendering the control sites undetectable, resulting in invalid results. The two-step method, where the sample first reacts with the antibody and then with signal molecules dissolved in the buffer solution, effectively washes away the whole blood sample from the channel, preventing background contamination. Therefore, the two-step method is feasible for whole blood samples, while the one-step method is not.

[0250] Example 7

[0251] Experimental objective: To compare and verify whether the one-step and two-step methods for detecting the barbed effect in IgE serum samples are present.

[0252] 1. Material preparation

[0253] The improved IgE microfluidic chip 3 (before improvement) and the improved two-step IgE microfluidic chip 4 (after improvement) differ in that the improved two-step microfluidic chip has an additional micro-sample dispensing port on the cover plate of the reaction zone channel 0.5 cm to the left of the control area. All of the above were manufactured by Shandong Mai Microbial Technology Co., Ltd.

[0254] IgE clinical serum sample S3 (concentration > 5000 ng / ml) was obtained from the relevant hospital;

[0255] Shandong Mai Microbial Technology Co., Ltd. produces fluorescence immunoassay analyzers, timers (such as stopwatches), and pipettes.

[0256] 2. Encapsulation site

[0257] The coating spot 1 is located at the detection site in the detection area and is coated with IgE antibody;

[0258] The second coating site is located at the quality control site in the detection area and is coated with secondary antibody.

[0259] The labeled region is fixed with dried IgE fluorescently labeled paired antibodies.

[0260] 3. Detection Method

[0261] 3.1 Improved IgE Two-Step Microfluidic Chip

[0262] The improved IgE two-step microfluidic chip was placed flat on the experimental stage. 10 μL of sample was added to the micro-sample wells, and after 5 seconds, the sample flowed backward through the control and detection sites. At this point, the absorbent material 4 was brought into contact with the channel to absorb excess sample until the flow stopped, allowing the sample remaining in the channel to react with the antibody for 90 seconds. 35 μL of sheep serum was added to the buffer well as a buffer solution. After the buffer solution mixed with the whole blood in the channel, the absorbent material 4 was used to absorb the water for 10 seconds. Once the remaining sample in the channel was absorbed and the signal molecules dissolved and filled the channel, the contact between the absorbent material 4 and the channel was disconnected. After reacting for 2 minutes, the absorbent material 4 was used again to absorb water for 2 minutes. The chip was then read using a fluorescence immunoassay analyzer, and the detection results of the IgE samples were recorded. Each sample was tested three times, and the signal values ​​of the control and detection sites were recorded for each sample.

[0263] 3.2 IgE microfluidic chip before improvement

[0264] Before the improvement, the IgE microfluidic chip was placed flat on the experimental stage. A 35 μL sample was added to the sample well of the microfluidic chip and left for 2 minutes. Water was absorbed using absorbent material 4 for 3 minutes, and then the chip was read by a fluorescence immunoassay analyzer. The detection results of the IgE sample were recorded. Each sample was tested three times, and the signal values ​​of the instrument's quality control site and detection site for each sample were recorded.

[0265] 3.3 Dilute the S3 sample 10-fold with sheep serum and repeat the test procedures in 3.1 (chip 6) and 3.2 (chip 5) to record the relevant data.

[0266] 4. Results

[0267] As shown in Table 7, comparing the detection results of chip 5 and chip 3, when using the one-step detection method, the peak value of the detection site decreased after the sample concentration increased by 10 times, and a bar appeared; comparing the detection results of chip 6 and chip 4, when using the two-step detection method, the peak value of the detection site increased with the sample concentration increased by 10 times, and no bar appeared.

[0268] Table 7 Sample detection results before and after microfluidic chip improvement

[0269] Experimental conclusion: The two-step method does not produce barbs when detecting high-concentration samples, while the one-step method does. The two-step method is more accurate than the one-step method.

[0270] Example 8

[0271] 1. Material preparation

[0272] The improved IgE microfluidic chip 7 (before improvement) and the improved two-step IgE microfluidic chip 8 (after improvement) differ in that the improved two-step microfluidic chip has an additional micro-sample dispensing port added to the cover plate on the reaction zone channel 0.5 cm to the left of the control area. All of the above were manufactured by Shandong Mai Microbial Technology Co., Ltd.

[0273] IgE clinical whole blood sample S1 was obtained from the relevant hospital;

[0274] Shandong Mai Microbial Technology Co., Ltd. produces fluorescence immunoassay analyzers, timers (such as stopwatches), and pipettes.

[0275] 2. Encapsulation site

[0276] The first coating point is located at detection site 322 in the detection area and is coated with IgE antibody.

[0277] The second coating site is located at the 322 quality control site in the detection area and is coated with a secondary antibody.

[0278] Labeled region 321 is fixed with dried IgE fluorescently labeled paired antibody.

[0279] 3. Detection Method

[0280] 3.1 Improved IgE Two-Step Microfluidic Chip

[0281] The improved IgE two-step microfluidic chip was placed flat on the experimental stage. 6 μL of sample was added to the micro-sample wells. After 30 seconds, 35 μL of sheep serum was added to the buffer well as a buffer solution. The buffer solution was then mixed with the whole blood in the channel, and absorbent material 4 was used to absorb the water for 10 seconds. Once the blood in the channel was absorbed and the signal molecules dissolved and filled the channel, the contact between absorbent material 4 and the channel was disconnected. After a 2-minute reaction, absorbent material 4 was used again for 2.5 minutes. The chip was then analyzed using a fluorescence immunoassay analyzer, and the detection results of the IgE samples were recorded. Each sample was tested three times, and the signal value at each detection site was recorded. The mean and deviation were calculated.

[0282] 3.2 IgE microfluidic chip before improvement

[0283] Before the improvement, the IgE microfluidic chip was placed flat on the experimental stage. 10 μL of sample was diluted approximately 10 times with diluent (containing sheep serum) and added to the sample well of the microfluidic chip. After 2 minutes, absorbent material 4 was used to absorb the water for 3 minutes. The chip was then read using a fluorescence immunoassay analyzer, and the detection results of the IgE sample were recorded. Each sample was tested three times, and the signal value of each detection site was recorded. The mean and deviation values ​​were calculated.

[0284] 4. Results

[0285] As shown in Table 8, the IgE detection results of the improved two-step IgE microfluidic chip 8 at 6 minutes showed a deviation of less than 3, while the IgE detection results of the unimproved IgE microfluidic chip 7 at 5 minutes showed a deviation of less than 7. Furthermore, the signal values ​​of the two-step method were higher than those of the one-step method. The deviation of the improved detection results was significantly smaller than that of the unimproved method. The different signal values ​​are due to the reaction sequence between the sample, antibody, and signal molecules, indicating that the improved chip provides more accurate detection results and a stronger signal.

[0286] Table 8 Sample detection results before and after microfluidic chip improvement

[0287] Example 9

[0288] 1. Material preparation

[0289] The improved quadruple-assay (RSV / FluA / FluB / COVID-19) microfluidic chip 9 and the improved quadruple-assay (test items as above) two-step microfluidic chip 10 differ in that the improved two-step microfluidic chip has an additional micro-sample application port on the cover of the reaction zone channel 0.5 cm to the left of the control area. Both were manufactured by Shandong Mai Microbial Technology Co., Ltd.

[0290] RSV clinical sample S1 was obtained from the relevant hospital;

[0291] Shandong Mai Microbial Technology Co., Ltd. produces fluorescence immunoassay analyzers, timers (such as stopwatches), and pipettes.

[0292] 2. Encapsulation site

[0293] The first coating site is located at detection site 1 (322 in the detection area) and is coated with RSV antibody; the second coating site is located at detection site 2 (322 in the detection area) and is coated with FluA antibody; the third coating site is located at detection site 3 (322 in the detection area) and is coated with FluB antibody; the fourth coating site is located at detection site 4 (322 in the detection area) and is coated with COVID-19 antibody; the fifth coating site is located at the quality control site (322 in the detection area) and is coated with secondary antibody.

[0294] The fluorescently labeled 321 region is immobilized with dried fluorescently labeled paired antibody.

[0295] 3. Detection Method

[0296] 3.1 Improved Quad-Measurement Two-Step Microfluidic Chip

[0297] The improved quadruple-step microfluidic chip was placed flat on the experimental stage. 35 μL of RSV sample was added to the micro-sample well. After 0 seconds, the RSV sample flowed backward through the control and detection sites. At this point, the absorbent material 4 was brought into contact with the channel to absorb excess sample until the flow stopped. The RSV sample remaining in the channel reacted with the antibody for 90 seconds. After the sample in the channel was absorbed and the signal molecules dissolved and filled the channel, the contact between the absorbent material 4 and the channel was disconnected. After a 2-minute reaction, the absorbent material 4 was used again to absorb water for 2.5 minutes. The chip was then analyzed using a fluorescence immunoassay analyzer, and the RSV sample detection results were recorded. Each sample was tested three times, and the signal value at detection site 1 for each sample was recorded. The mean and deviation values ​​were calculated.

[0298] 3.2 Improved Quadruple Microfluidic Chip

[0299] Before the improvement, the four-cell microfluidic chip was placed flat on the experimental stage. 35 μL of RSV sample was added to the sample well of the microfluidic chip and waited for 2 minutes to allow the sample to react with the antibody within the channel. Then, absorbent material 4 was used to absorb water for 3 minutes. The chip was then read using a fluorescence immunoassay analyzer, and the detection results of the RSV sample were recorded. Each sample was tested three times, and the signal value of detection site 1 for each sample was recorded. The mean and deviation values ​​were calculated.

[0300] 4. Results

[0301] As shown in Table 9, the RSV detection results of the improved quadruple assay two-step microfluidic chip 10 at 6 minutes showed a deviation of less than 3, while the RSV detection results of the original quadruple assay microfluidic chip 9 at 5 minutes showed a deviation of less than 4. Furthermore, the signal values ​​of the two-step method were higher than those of the one-step method. The deviation of the improved detection results was smaller than that of the original. The different signal values ​​are due to the reaction sequence between the sample, antibody, and signal molecules, indicating that the improved chip provides more accurate detection results and a stronger signal.

[0302] Table 9 Sample detection results before and after microfluidic chip improvement

[0303] The above technical solutions only embody preferred technical solutions of the present invention. However, those skilled in the art will understand that various modifications and alterations can be made to the above specific embodiments without departing from the concept of the present invention. Furthermore, various combinations of the technical features and structures proposed in various aspects of the present invention can be made without exceeding the protection scope of the present invention, which is determined by the appended claims.

[0304] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0305] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0306] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

Claims

1. A microfluidic chip, comprising: Substrate (1); Cover (2), including a groove (21) extending along the length direction (L); as well as The microchannel (3) is formed by the substrate (1) and the groove (21). The feature is that the groove (21) of the cover plate (2) includes a channel area (211), and the channel area (211) is provided with a sample application area (2111), a reaction area (2112), and a control area (2113) in sequence along the length direction (L). The sample application area (2111), the reaction area (2112), and the control area (2113) are in fluid communication with each other. The control zone (2113) is equipped with a control valve (2113a), which is configured to control the flow of liquid from the reaction zone (2112) to the control zone (2113). The height of the reaction zone (2112) is lower than the height of the control zone (2113), and the width of the reaction zone (2112) is smaller than the width of the control zone (2113).

2. The microfluidic chip according to claim 1, characterized in that, The control zone (2113) gradually narrows from the middle to the reaction zone (2112) to make it easier to contact the liquid in the reaction zone and maintain the detached state.

3. The microfluidic chip according to claim 1, characterized in that, The control valve (2113a) includes: A movable fluid suction device (2113a1) capable of contacting or moving away from the reaction zone (2112); and Braking hole (2113a3) is provided on the side away from the reaction zone (2112). The braking hole (2113a3) is a rectangular through hole that penetrates the substrate (1) and the cover plate (2). The fluid suction member (2113a1) is provided with a through hole (4) at the end away from the reaction zone. The through hole (4) is inside the braking hole (2113a3).

4. The microfluidic chip according to claim 3, characterized in that, The control area (2113) is provided with a locking part (5), which is configured to hold the fluid suction member (2113a1) away from the reaction area (2112).

5. The microfluidic chip according to claim 4, characterized in that, The locking portion (5) includes a protrusion arranged in the width direction (W), and the fluid suction member (2113a1) is correspondingly provided with a recess that mates with the protrusion.

6. The microfluidic chip according to claim 3, characterized in that, The cover plate (2) is provided with a retention portion (2113a2) at the connection between the control zone (2113) and the reaction zone (2112). The retention portion (2113a2) extends at least partially into the reaction zone (2112), such that when the fluid suction member (2113a1) contacts the channel of the reaction zone (2112), at least a portion of the fluid suction member (2113a1) is inserted into the retention portion (2113a2).

7. The microfluidic chip according to claim 6, characterized in that, The retention part (2113a2) is a toothed part, which is constructed as a concave arc shape extending into the reaction zone (2112). The lower end of the arc shape is provided with a sawtooth or wave structure. The fluid suction member (2113a1) gradually narrows on the side near the reaction zone (2112) and has a notch in the middle on the side near the reaction zone (2112). When the fluid suction member (2113a1) contacts the channel of the reaction zone (2112), the notch is inserted into the toothed part (2113a2).

8. The microfluidic chip according to claim 6, characterized in that, The retention section (2113a2) is an inclined groove that slopes toward the reaction zone (2112).

9. The microfluidic chip according to any one of claims 3 to 8, characterized in that, The fluid suction element (2113a1) includes a water-absorbing material, wherein the water-absorbing material includes at least one of a desiccant, an antioxidant, and a moisture indicator.

10. The microfluidic chip according to claim 1, characterized in that, The sample application area (2111) is provided with a sample application hole that connects the upper and lower surfaces of the cover plate (2). The height of the sample application hole on the lower surface of the cover plate (2) is on the same horizontal plane as the height of the reaction area (2112).

11. The microfluidic chip according to claim 1, characterized in that, The groove (21) is rectangular, and the shape of the substrate (1) matches the groove (21). The substrate (1) is placed in the groove (21) and is on the same horizontal plane as the surface of the cover plate (2).

12. The microfluidic chip according to claim 1, characterized in that, The groove (21) of the cover plate (2) is provided with a channel area (211), a barrier area (212) and a pressing area (213) from the middle to the outside along the width direction (W). The height of the channel area (211) and the barrier area (212) is higher than the height of the pressing area (213), so that the substrate (1) is placed in the groove (21) on the lower surface of the cover plate (2) and contacts the pressing area (213), and a gap is formed between the substrate (1) and the channel area (211) and the barrier area (212).

13. The microfluidic chip according to claim 12, characterized in that, The barrier region (212) is located on the periphery of the sample application area (2111) and / or the reaction area (2112) of the channel area (211), and in the length direction (L), the barrier region (212) covers at least part of the reaction area (2112), or covers the sample application area (2111) and / or the reaction area (2112).

14. The microfluidic chip according to claim 12, characterized in that, in, The substrate (1) is coated with a hydrophobic material at the position of the barrier area (212) corresponding to the groove (21) of the cover plate (2).

15. The microfluidic chip according to claim 14, characterized in that, The reaction zone (2112) and the barrier zone (212) are on the same horizontal plane, and the barrier zone (212) is also coated with a hydrophobic material.

16. The microfluidic chip according to claim 12, characterized in that, in, The substrate (1) is laser-processed at the position of the barrier area (212) corresponding to the groove (21) of the cover plate (2) to form a hydrophobic surface.

17. The microfluidic chip according to claim 14 or 16, characterized in that, The reaction zone (2112) and the barrier zone (212) are on the same horizontal plane, and the barrier zone (212) is laser-treated to form a hydrophobic surface.

18. The microfluidic chip according to claim 14, characterized in that, The height of the reaction zone (2112) is lower than the height of the barrier zone (212), so that the sample will not enter the barrier zone (212) due to gravity when it flows along the reaction zone (2112).

19. A microfluidic chip, comprising: Substrate (1); Cover slip (2); as well as The microchannel (3) is formed by the substrate (1) and the cover plate (2). The microchannel (3) is characterized in that it comprises a buffer loading area (31), a reaction area (32), and a control area (33), wherein the buffer loading area (31), the reaction area (32), and the control area (33) are sequentially connected along the length direction (L), and a movable absorbent material (4) is disposed within the control area (33), wherein the absorbent material (4) can contact or move away from the reaction area (32), and The reaction zone (32) includes a labeling zone (321), a detection zone (322), and a sample loading zone (323), with the detection zone (322) and the sample loading zone (323) both located on the same side of the labeling zone (321).

20. The microfluidic chip according to claim 19, characterized in that, The cover plate (2) has a groove along its length (L), and the groove and the substrate (1) together form the microchannel (3).

21. The microfluidic chip according to claim 19, characterized in that, The detection area (322) is provided with detection sites and quality control sites in sequence along the length direction (L), and the number of detection sites and quality control sites is one or more.

22. The microfluidic chip according to claim 19, characterized in that, The labeled region (321) includes an antibody / antigen labeled with a signal molecule. The detection site of the detection region (322) is coated with an antibody / antigen that can bind to the target analyte. The target analyte can bind to the antibody / antigen of the labeled region (321) and the detection site simultaneously to form a double antigen / antibody sandwich complex. The quality control site of the detection region (322) is coated with an antibody / antigen that can bind to the antibody / antigen labeled with the signal molecule of the labeled region (321).

23. The microfluidic chip according to claim 19, characterized in that, A braking hole (331) is provided on the side of the control area (33) opposite to the reaction area (32). The braking hole (331) is a rectangular through hole that penetrates the substrate (1) and the cover plate (2) and extends at least partially into the water-absorbing material (4) in the control area (33). The water-absorbing material (4) has a through hole at the end away from the reaction area, and the through hole is located in the braking hole (331).

24. The microfluidic chip according to claim 19, characterized in that, The buffer solution loading area (31) is provided with a buffer solution hole that connects the upper and lower surfaces of the cover plate (2), and the sample loading area (323) is provided with a sample loading hole that connects the upper and lower surfaces of the cover plate (2).

25. The microfluidic chip according to claim 19, characterized in that, The sample application area (323) and the labeling area (321) are respectively located on both sides of the detection area (322).

26. The microfluidic chip according to claim 19, characterized in that, The sample application area (323) is located between the marking area (321) and the detection area (322).

27. The microfluidic chip according to claim 22, characterized in that, The antigen / antibody labeled with the signal molecule is an antigen / antibody labeled with the signal molecule.

28. A method for detecting a microfluidic chip, characterized in that, The method of using a microfluidic chip as described in any one of claims 1 to 9 for detection includes at least the following steps: The sample is added into the microchannel (3) through the sample addition area (323), and the absorbent material (4) is kept away from the reaction area (32); After the sample flows backward through the detection zone (322) under capillary action and meets the first judgment condition, the absorbent material (4) is moved to contact the reaction zone (32) to absorb the excess sample until the flow is interrupted, and the sample remaining in the microchannel (3) reacts with the detection zone (322); then, the absorbent material (4) detaches from or continues to contact the reaction zone (32); After the second reaction time period, the buffer solution is added into the microchannel (3) through the buffer solution loading area (31). The buffer solution passes through the labeling area (321) under capillary action, washing away the antigen / antibody coated with the signal molecules in the labeling area (321) and entering the detection area (322). When the signal molecules in the channel of the reaction area (32) merge with the sample, the absorbent material (4) in the moving control area (33) contacts the reaction area (32), and the excess sample is adsorbed into the control area (33) through the absorbent material (4). After the excess sample in the channel is absorbed by the absorbent material (4) and the signal molecules in the labeled area (321) are dissolved and spread throughout the reaction area (32) channel, the absorbent material (4) is moved away from the reaction area (32) so that the absorbent material (4) breaks the contact with the reaction area (32) channel. After the third reaction time period, the absorbent material (4) in the moving control area (33) comes into contact with the detection area (322) and adsorbs the excess liquid, including signal molecules, into the control area (33) through the absorbent material (4); Measure the signal value of the detection area (322).

29. The detection method according to claim 28, characterized in that, The first judgment condition includes: the signal intensity of the signal molecules in the detection area (322) is greater than or equal to the threshold after the first time period or the amount of sample liquid is greater than or equal to the predetermined amount.

30. The detection method according to claim 29, characterized in that, The first time period is in the range of 0 to 60 seconds, and the predetermined amount is in the range of 15 μL to 50 μL.

31. The detection method according to claim 28 or 29, characterized in that, The second time period is in the range of 10 seconds to 300 seconds, and the third time period is in the range of 30 seconds to 5 minutes.

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